(a) Field of the Invention
The invention relates to a novel gene, CaKRE9, isolated in the yeast pathogen, Candida albicans, that is a functional homolog of the S. cerevisiae KRE9 gene and which is essential for cell wall glucan synthesis, and to novel antifungal screening assays.
(b) Description of Prior Art
Fungi constitute a vital part of our ecosystem but once they penetrate the human body and start spreading they cause infections or xe2x80x9cmycosisxe2x80x9d and they can pose a serious threat to human health. Fungal is infections have dramatically increased in the last 2 decades with the development of more sophisticated medical interventions and are becoming a significant cause of morbidity and mortality. Infections due to pathogenic fungi are frequently acquired by debilitated patients with depressed cell-mediated immunity such as those with human immunodeficiency virus (HIV) and now also constitute a common complication of many medical and surgical therapies. Risk factors that predispose individuals to the development of mycosis include neutropenia, use of immunosuppressive agents at the time of organ transplants, intensive chemotherapy and irradiation for hematopoietic malignancies or solid tumors, use of corticosteroids, extensive surgery and prosthetic devices, indwelling venous catheters, hyperalimentation and intravenous drug use, and when the delicate balance of the normal flora is altered through antimicrobial therapy.
The yeast genus Candida constitutes one of the major groups that cause systemic fungal infections and the five medically relevant species which are most often recovered from patients are C. albicans, C. tropicalis, C. glabrata, C. parapsilosis and C. krusei. 
Much of the structure of fungal and animal cells along with their physiology and metabolism is highly conserved. This conservation in cellular function has made it difficult to find agents that selectively discriminate between pathogenic fungi and their human hosts, in the way that antibiotics do between bacteria and man. Because of this, the common antifungal drugs, like amphotericin B and the azole-based compounds are often of limited efficacy and are frequently highly toxic. In spite of these drawbacks, early initiation of antifungal therapy is crucial in increasing the survival rate of patients with disseminated candidiasis. Moreover, resistance to antifungal drugs is becoming more and more prominent. For example, 6 years after the introduction of fluconazole, an alarming proportion of Candida strains isolated from infected patients have been found to be resistant to this drug and this is especially the case with vaginal infections. There is thus, a real and urgent need for specific antifungal drugs to treat mycosis.
The Fungal Cell Wall: a Resource for New Antifungal Targets
In recent years, we have focused our attention on the fungal extracellular matrix, where the cell wall constitutes an essential, fungi-specific organelle that is absent from human/mammalian cells, and hence offers an excellent potential target for specific antifungal antibiotics. The cell wall of fungi is essential not only in maintaining the osmotic integrity of the fungal cell but also in cell growth, division and morphology.
The cell wall contains a range of polysaccharide polymers, including chitin, xcex2-glucans and O- and N-linked mannose sidechains of glycoproteins. xcex2-glucans, homopolymers of glucose, are the main structural component of the yeast cell wall, and constitute up to 60% of the dry weight of the cell wall. Based on their chemical linkage, two different types of polymers can be found: xcex21,3-glucan and xcex21,6-glucan. The xcex21,3-glucan is the most abundant component of the cell wall and it contains on average 1500 glucose residues per molecule. It is mainly a linear molecule but contains some 1,6-linked branchpoints. The xcex21,6-glucan is a smaller and highly branched molecule comprised largely of 1,6-linked glucose residues with a small proportion of 1,3-linked residues. The average size of xcex21,6-glucan is approximately 400 residues per molecule. The xcex21,6-glucan polymer is essential for cell viability as it acts as the xe2x80x9cgluexe2x80x9d covalently linking glycoproteins and the cell wall polymers xcex21,3-glucan and chitin together in a crosslinked extracellular matrix.
It would be highly desirable to be provided with the identification and subsequent validation of new cell wall related targets that can be used in specific enzymatic and cellular assays leading to the discovery of new clinically useful antifungal compounds.
One aim of the present invention is to provide the identification and subsequent validation of a new target that can be used in specific enzymatic and cellular assays leading to the discovery of new clinically useful antifungal compounds.
Although a gene involved in the cellular growth of S. cerevisiae was identified, there are no certainties that there would be a homolog in Candida albicans or if present that it would have the same function.
In accordance with the present invention a gene was isolated, CaKRE9, in the yeast pathogen, Candida albicans, that is a functional homolog of the S. cerevisiae KRE9 gene and which is essential for cell wall glucan synthesis. The gene is not found in humans and when it is inactivated in C. albicans, the cell cannot survive when grown on glucose, thus, validating it as a wholly new target for antifungal drug discovery.
Using the gene of the present invention, we intend to utilize novel drug screening assays for which we possess all the genetic tools.
In accordance with the present invention there is provided an isolated DNA which codes for a gene essential for cell wall glucan synthesis of Candida albicans, wherein the gene is referred to as CaKRE9, wherein the sequence of the DNA is as set forth in FIG. 1.
In accordance with the present invention there is also provided an antifungal screening assay for identifying a compound which inhibits the synthesis, assembly and/or regulation of xcex21,6-glucan, which comprises the steps of:
a) synthesizing xcex21,6-glucans in vitro from activated sugar monomer/polymer and specific xcex21,6-glucan synthetic proteins;
b) subjecting step a) to a high throughput compound screen determining absence or presence of xcex21,6-glucan, wherein absence of xcex21,6-glucan is indicative of an antifungal compound.
In accordance with the present invention there is also provided an in vivo antifungal screening assay for identifying compounds which inhibit the synthesis, assembly and/or regulation of xcex21,6-glucan, which comprises the steps of:
a) separately cultivating a mutant yeast strain lacking one gene for synthesis of xcex21,6-glucans and a wild type yeast strain with activated sugar monomer/polymer UDP-glucose;
b) subjecting both yeast strains of step a) to the screened compound and determining if the compound selectively inhibits growth of wild type strain which is indicative of an antifungal compound.
In accordance with the present invention there is also provided an in vitro method for the diagnosis of diseases caused by fungal infection in a patient, which comprises the steps of:
a) obtaining a biological sample from the patient;
b) subjecting the sample to PCR using a primer pair specific for CaKRE9 gene, wherein a presence of the gene is indicative of the presence of fungal infection.
In accordance with the present invention, the gene is CaKRE9.
In accordance with the present invention there is also provided an in vitro method for the diagnosis of diseases caused by fungal infection in a patient, which comprises the steps of:
a) obtaining a biological sample from the patient;
b) subjecting the sample to an antibody specific for CaKre9p antigen, wherein a presence of the antigen is indicative of the presence of fungal infection.
In accordance with one embodiment of the present invention, the fungal infection may be caused by Candida.
In accordance with the present invention there is also provided the use of at least one of KRE9 and CaKre9 nucleic acid sequences and fragments thereof as a probe for the isolation of KRE9 homologs in all fungi.
For the purpose of the present invention the following terms are defined below.
The term a xe2x80x9cmutant yeast strainxe2x80x9d is intended to mean any yeast strain lacking one gene for synthesis of xcex21,6-glucan, such as KRE9 and homologs thereof.
The term a xe2x80x9cwild type yeast strainxe2x80x9d is intended to mean any yeast strain containing the KRE9 gene or a homolog thereof or a plasmid overexpressing the KRE9 gene or a homolog thereof.